Despite their broad medical importance as dominant mechanisms underlying the rapid dissemination of antibiotic resistance and infection by many bacterial pathogens, there is a fundamental lack of mechanistic understanding how type IV secretion systems (T4SSs) translocate macromolecular substrates to bacterial or eukaryotic target cells. The VirB/VirD4 system of Agrobacterium tumefaciens and closely-related plasmid conjugation systems in Escherichia coli serve as important models for detailed structure-function studies of T4SSs. The long-term goal of work in this laboratory is to describe in complete molecular terms the T4SS machine biogenesis pathway, the basis for substrate recognition and transfer across the cell envelope, and the nature of the machine - target cell contact. New structural information has shown that the T4SSs of Gram- negative bacteria are composed of a large outer membrane-associated complex (OMC; also designated as the core complex) and an equally large inner membrane complex (IMC), with a narrow stalk connecting the two substructures. This structure raises new questions about the substrate secretion pathway and the mechanisms mediating assembly of conjugative pili. We hypothesize that T4SSs function either as substrate channels or as pilus biogenesis systems and that a combination of intra- and extracellular signals regulate these alternative outcomes. We propose two specific aims: 1) Define the nature of substrate-receptor docking and the contributions of channel ATPases to substrate processing and delivery across the inner membrane and ii) Define the pilus biogenesis pathway and the regulatory checkpoints governing substrate transfer and pilus nucleation and extension across the outer membrane. For Aim 1, recent findings drive our refined studies exploring the roles of translocation signals in the substrate - receptor binding, as well as experiments testing the contributions of two ATPases to chaperone release and substrate unfolding prior to translocation. For Aim 2, recent structural advances and biochemical findings support tests of specific models for pilus nucleation at the IMC and extension through the central chamber of the OMC. A combination of intracellular signals, including substrate binding and ATP energy, is postulated to induce conformational changes in the OMC for activation of substrate transfer at the expense of pilus production. These studies interface with ongoing collaborative efforts to obtain high-resolution T4SS structures and donor-target cell mating junctions. The work is innovative, in concept by exploiting a highly efficient conjugation machine that we have now shown to translocate effector proteins, and in approach through incorporation of state-of-the art in vivo assays to define dynamic steps in substrate translocation and pilus assembly. The work is expected to stimulate new basic research directions and provide a critical knowledge base for applied studies aimed at suppressing antibiotic resistance spread and disease progression by many clinically significant pathogens.